Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription

• US 10,000,772 B2
• Filed: 04/13/2015
• Issued: 06/19/2018
• Est. Priority Date: 05/25/2012
• Status: Active Grant

• Current Assignee: The University Of Vienna, Regents of the University of California (University of California)
• Original Assignee: Emmanuelle Charpentier, The University Of Vienna, Regents of the University of California (University of California)
• Inventors: Doudna, Jennifer A., Jinek, Martin, Charpentier, Emmanuelle, Chylinski, Krzysztof
• Primary Examiner(s): Hammell, Neil P
• Application Number:
  US14/685,502
• Publication Number:
  US 20160130608A1
• Time in Patent Office: 1,163 Days
• Field of Search: None
• CPC Class Codes: A01H 6/4684 Zea mays [maize]
• CPC Class Codes: A01K 67/027 New or modified breeds of vertebrates
• CPC Class Codes: A61K 38/465 acting on ester bonds (3.1), e.g. lipases, ribonucleases
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 15/746 for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)
• CPC Class Codes: C12N 15/90 Stable introduction of foreign DNA into chromosome
• CPC Class Codes: C12N 15/902 using homologous recombination
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/13 Decoys
• CPC Class Codes: C12N 2310/14 : interfering N.A.
• CPC Class Codes: C12N 2310/20 : involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 2310/31 : of the backbone
• CPC Class Codes: C12N 2310/32 : of the sugar
• CPC Class Codes: C12N 2310/33 : of the base
• CPC Class Codes: C12N 2310/3519 : Fusion with another nucleic acid
• CPC Class Codes: C12N 2310/531 : Stem-loop; Hairpin
• CPC Class Codes: C12N 2800/80 : Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
• CPC Class Codes: C12N 9/22 : Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Q 1/686 : Polymerase chain reaction [PCR]
• CPC Class Codes: C12Y 301/04 : Phosphoric diester hydrolases (3.1.4)
• CPC Class Codes: H01L 21/02312 : treatment by exposure to a gas or vapour
• CPC Class Codes: H01L 21/02365 : Forming inorganic semiconducting materials on a substrate for light-sensitive devices H01L31/00
• CPC Class Codes: H01L 33/0075 : comprising nitride compounds
• CPC Class Codes: Y02A 50/30 : Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The present disclosure provides a DNA-targeting RNA that comprises a targeting sequence and, together with a modifying polypeptide, provides for site-specific modification of a target DNA and/or a polypeptide associated with the target DNA. The present disclosure further provides site-specific modifying polypeptides. The present disclosure further provides methods of site-specific modification of a target DNA and/or a polypeptide associated with the target DNA The present disclosure provides methods of modulating transcription of a target nucleic acid in a target cell, generally involving contacting the target nucleic acid with an enzymatically inactive Cas9 polypeptide and a DNA-targeting RNA. Kits and compositions for carrying out the methods are also provided. The present disclosure provides genetically modified cells that produce Cas9; and Cas9 transgenic non-human multicellular organisms.

Claims

1. A method of modifying a target DNA molecule, the method comprising:

contacting a target DNA molecule having a target sequence with a complex comprising;

(a) a Cas9 protein; and

(b) a DNA-targeting RNA comprising;

(i) a targeter-RNA that hybridizes with the target sequence, and

(ii) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA (dsRNA) duplex of a protein-binding segment,

wherein the activator-RNA hybridizes with the targeter-RNA to form a total of 10 to 15 base pairs,

wherein said contacting takes place outside of a bacterial cell and outside of an archaeal cell,

thereby resulting in modification of the target DNA molecule.

2. The method of claim 1, wherein said modification of the target DNA molecule is cleavage of the target DNA molecule.

3. The method of claim 1, wherein the target sequence is 15 nucleotides (nt) to 18 nt long.

4. The method of claim 1, wherein the target sequence is 18 nucleotides (nt) to 25 nt long.

5. The method of claim 1, wherein the target DNA molecule is chromosomal DNA.

6. The method of claim 1, wherein the activator-RNA comprises the 26 nucleotide tracrRNA sequence set forth in SEQ ID NO:

441.

7. The method of claim 1, wherein the targeter-RNA and/or the activator-RNA comprises one or more of:

a non-natural internucleoside linkage, a nucleic acid mimetic, a modified sugar moiety, and a modified nucleobase.

8. The method of claim 1, wherein the targeter-RNA and/or the activator-RNA comprises one or more of:

(i) a non-natural internucleoside linkage selected from a phosphorothioate, an inverted polarity linkage, and an abasic nucleoside linkage;

(ii) a locked nucleic acid (LNA); and

(iii) a modified sugar moiety selected from 2′

-O-methoxyethyl, 2′

-O-methyl, and 2′

-fluoro.

9. The method of claim 1, wherein the targeter-RNA and/or the activator-RNA comprises one or more of:

a peptide nucleic acid (PNA), a morpholino nucleic acid, a cyclohexenyl nucleic acid (CeNA), and/or a locked nucleic acid (LNA).

10. The method of claim 1, wherein the targeter-RNA and/or the activator-RNA comprises one or more modified sugar moieties selected from:

2′

-O-(2-methoxyethyl), 2′

-dimethylaminooxyethoxy, 2′

-dimethylaminoethoxyethoxy, 2′

-O-methyl, and 2′

-fluoro.

11. The method of claim 1, wherein the targeter-RNA and/or the activator-RNA is conjugated to a moiety selected from:

a polyamine;

a polyamide;

a polyethylene glycol;

a polyether;

a cholesterol moiety;

a cholic acid;

a thioether;

a thiocholesterol;

an aliphatic chain;

a phospholipid;

an adamantane acetic acid;

a palmityl moiety;

an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety;

a biotin;

a phenazine;

a folate;

a phenanthridine;

an anthraquinone;

an acridine;

a fluorescein;

a rhodamine;

a fluor; and

a coumarin.